GLIPR1 Tumor Suppressor Gene Expressed by Adenoviral Vector As

Published OnlineFirst September 20, 2011; DOI: 10.1158/1078-0432.CCR-11-1899

Clinical Cancer Cancer Therapy: Clinical Research

GLIPR1 Tumor Suppressor Gene Expressed by Adenoviral Vector as Neoadjuvant Intraprostatic Injection for Localized Intermediate or High-Risk Prostate Cancer Preceding Radical Prostatectomy

Guru Sonpavde1,6, Timothy C. Thompson7, Rajul K. Jain3, Gustavo E. Ayala2, Shinji Kurosaka7, Kohei Edamura7, Ken-ichi Tabata7, Chengzhen Ren7, Alexei A. Goltsov7, Martha P. Mims1, Teresa G. Hayes1,6, Michael M. Ittmann5, Thomas M. Wheeler2, Adrian Gee3, Brian J. Miles4, and Dov Kadmon4

Abstract Background: GLIPR1 is upregulated by p53 in prostate cancer cells and has preclinical antitumor activity. A phase I clinical trial was conducted to evaluate the safety and activity of the neoadjuvant intraprostatic injection of GLIPR1 expressing adenovirus for intermediate or high-risk localized prostate cancer before radical prostatectomy (RP). Methods: Eligible men had localized prostate cancer (T1-T2c) with Gleason score greater than or equal to 7 or prostate-speciﬁc antigen 10 ng/mL or more and were candidates for RP. Patients received the adenoviral vector expressing the GLIPR1 gene by a single injection into the prostate followed four weeks later by RP. Six viral particle (vp) dose levels were evaluated: 1010,5 1010,1011,5 1011,1012, and 5 1012 vp. Results: Nineteen patients with a median age of 64 years were recruited. Nine men had T1c, 4 had T2a, and 3 had T2b and T2c clinical stage. Toxicities included urinary tract infection (n ¼ 3), ﬂu-like syndrome (n ¼ 3), fever (n ¼ 1), dysuria (n ¼ 1), and photophobia (n ¼ 1). Laboratory toxicities were grade 1 elevated AST/ALT (n ¼ 1) and elevations of PTT (n ¼ 3, with 1 proven to be lupus anticoagulant). No pathologic complete remission was seen. Morphologic cytotoxic activity, induction of apoptosis, and nuclear p27Kip1 þ þ þ upregulation were observed. Peripheral blood CD8 , CD4 , and CD3 T-lymphocytes were increased, with upregulation of their HLA-DR expression and elevations of serum IL-12. Conclusions: The intraprostatic administration of GLIPR1 tumor suppressor gene expressed by an adenoviral vector was safe in men, with localized intermediate or high-risk prostate cancer preceding RP. Preliminary evidence of biologic antitumor activity and systemic immune response was documented. Clin Cancer Res; 17(22); 1–9. 2011 AACR.

Introduction of prostate-speciﬁc antigen (PSA) screening and radical prostatectomy (RP) can improve outcomes in appropriately Prostate cancer is the most common solid tumor diag- selected patients. Androgen deprivation therapy (ADT) is nosed in the United States with approximately 200,000 the conventional frontline systemic therapy for hormone- cases and 30,000 deaths in a year (1). Prostate cancer is naive prostate cancer. However, ADT has deleterious effects commonly detected when still localized because the advent on quality of life and bone health, and progression to castration-resistant prostate cancer (CRPC) is almost unavoidable in men with advanced disease. Treatment Authors' Afﬁliations: 1Department of Medicine, Section of Medical Oncol- ogy, Departments of 2Pathology and 3Pediatrics and Medicine, and 4Scott options are limited in patients with metastatic CRPC, and Department of Urology, Baylor College of Medicine; 5Department of chemotherapy (docetaxel, cabazitaxel), immunotherapy 6 Pathology, Michael E. DeBakey Veterans Affairs Medical Center; and (sipuleucel-T), and androgen synthesis inhibitors (abirater- 7Department of Genitourinary Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas one acetate) have shown modest extensions of survival (2–6). Clearly, there is a need for more effective and Note: This work was presented in part as a poster at the Genitourinary Cancer Symposium, March 2010, San Francisco, CA. tolerable agents for prostate cancer. Corresponding Author: Dov Kadmon, Scott Department of Urology, Given that the vast majority of men present with Baylor College of Medicine, 6620 Main St., Suite 1325, Houston, TX 77030. localized disease, the paradigm of neoadjuvant therapy E-mail: [email protected] preceding RP may improve outcomes and facilitate the doi: 10.1158/1078-0432.CCR-11-1899 developmentofnewagentsforprostatecancerbypro- 2011 American Association for Cancer Research. viding an early signal of activity (7). The evaluation of

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Materials and Methods Translational Relevance Patient eligibility The intraprostatic administration of a single injection Patients were required to have clinical stage T1c– of GLIPR1, a p53-regulated tumor suppressor gene, T2cN0M0 adenocarcinoma of the prostate with a Gleason expressed by an adenoviral vector was safe and showed score 7 or PSA 10 ng/mL. All participants had to have a morphologic cytotoxicity, biologic antitumor activity needle biopsy of the prostate (at least 12 cores) to obtain (upregulation of apoptosis and a cyclin-dependent tissue for pathologic analysis. A baseline chest X-ray, bone kinase inhibitor), and systemic immune responses scan, and CT scan of the abdomen and pelvis were man- (increased activated T lymphocytes and IL-12) in men dated for staging. Patients were also required to be candi- with localized intermediate and high-risk prostate can- dates for RP. Written informed consent was obtained from cer preceding prostatectomy. No dose limiting toxicities all of the patients. were observed, suggesting that higher or more frequent doses may warrant evaluation. Given the excellent tol- Construction of adenoviral vector GLIPR1 erability profile in this elderly population with frequent Clinical grade replication-defective Ad5GLIPR1 viral vec- comorbidities, the further development of this thera- tor was prepared in the Baylor College of Medicine (BCM) peutic modality, both alone and in combination Cell and Gene Therapy GMP facility. Human GLIPR1 cDNA approaches, may be justified. was PCR amplified from pcDNA3-hRTVP1 vector with specific primers that contained sequence for XbaI and KpnI restriction enzymes (underlined), respectively (upper primer: 50-CTAGTCTAGAGCCACCATGCGTGTCACACTT- neoadjuvant therapy is justified in high-risk cases defined GCT-30, lower primer: 50-GGGGTACCTTAGTCCAAAA- by high PSA, Gleason score, and clinical stage. Because GAACTAA-30). In addition, upstream primer contained pathologic and biological activity can be rapidly deter- optimized Kozak sequence (GCCACC, bold font) in front mined after surgery, the efficacy of a systemic regimen is of ATG codon of hRTVP-1 cDNA. The resulting 821 bp PCR evident with a relatively small number of patients before fragment containing complete coding sequence of GLIPR1 long-term follow-up. Neoadjuvant ADT followed by RP was cloned into XbaI and KpnI sites of pShutlle-X (Clontech) improves pathologic outcomes with no definitive evi- transfer vector and sequenced. The coding sequence for dence for improvement in long-term clinical outcomes GLIPR1 cDNA was not modified. Three recombinant pShut- (8). Ongoing phase III trials are evaluating the impact of tle-hRTVPk vector clones were analyzed for hRTVP-1 expres- combination chemotherapy and ADT on outcomes based sion by Western blot after transient transfection into A459 on the suggestion of improved pathologic outcomes (9). cell line. All 3 pShuttle-hRTVPk clones with modified Kozak In addition, biologic and chemotherapeutic agents have sequence showed better expression of hRTVP-1 protein been evaluated without concomitant ADT to obtain a compared with the original pShuttle-hRTVP-1 vector signal of activity and provide proof of concept (10–13). (Fig. 1). Clone pShuttle-hRTVPk1 was used for construction The intraprostatic delivery of genes by employing a vector of the adenoviral vector. has also been studied (14–16). Clontech adeno-X expression system protocol was used Because of the established association between loss of and the I-Ceu/PI-SceI fragment containing expression cas- p53 function and prostate cancer metastasis, we have pur- sette CMV-hRTVPk1-polyA was used to generate the sued the identification, characterization, and functional analysis of p53 target genes in prostate cancer (17–19). We identified the GLIPR1 (glioma pathogenesis related protein), previously termed RTVP-1 (related to testes specific, GLIPR1 vespid, and pathogenesis proteins), mRNA as being upregulated by p53 in mouse prostate cancer cells. Both mouse and human GLIPR1 contain p53 binding elements in β-Actin promoter and intronic sequences. GLIPR1 was shown to have proapoptotic, antiangiogenic, immunostimulatory, 123456 and metastasis-suppressing activity (20). Adenoviral vector-mediated GLIPR1delivery in vivo was capable of eradi- fi cating micrometastatic disease (21, 22). GLIPR1 is down- Figure 1. Modi cation of Kozak sequence of GLIPR1/RTVP-1 cDNA improves GLIPR1 protein expression. Unmodified and modified GLIPR1/ regulated, in part, by gene methylation in prostate cancer RTVP-1 cDNA constructs were transfected in A459 cell line and cell compared with normal prostate tissue (23). Given that lysates were analyzed by Western blot for GLIPR1 protein expression GLIPR1 may confer antitumor activity, a phase I clinical levels 48 hours after transfection. Lanes: 1, pcDNA3.1 (control vector); 2, trial was conducted to evaluate the safety and biologic pShuttle-hRTVP-1: 3, pSecTag-hRTVP-1; 4, pShuttle-hRTVPk1, 5, activity of adenovirus delivered in situ GLIPR1 gene therapy pShuttle-hRTVPk2, 6, pShuttle-hRTVPk3. All 3 pShuttle-hRTVPk clones with modified Kozak sequence (lanes 4–6) showed increased expression for localized intermediate and high-risk prostate cancer of GLIPR1/hRTVP-1 protein compared with the original pShuttle-hRTVP- before RP. 1 vector (lane 2).

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adenoviral vector. The construction of Ad5hRTVPk1 was blood cells were lysed with formic acid and the samples confirmed by restriction endonuclease mapping and by fixed with paraformaldehyde using a Coulter Q-Prep work- conventional dideoxy nucleotide sequencing. Expression station. Forward and side scatter were set to distinguish of RTVP-1 was confirmed by Western blot. For the produc- lymphocyte, macrophage, and granulocyte populations tion of recombinant adenoviruses, 293 cells were trans- from debris with a BD FACSCalibur Flow Cytometer (Bec- fected with Ad5hRTVPk1. Once cytopathic effects were ton Dickinson and Company). Results were expressed as the observed, cells were harvested and lysed. This lysate was percent of cells in the lymphocyte gate. used for plaque purification. Further production and characterization of a clinical grade Ad5hRTVP-1 vector was done Tumor tissue, blood- and plasma-based correlative by BCM Cell and Gene Therapy GMP facility. Comprehen- studies sive information about the clinical grade vector was pro- Tumor tissue was examined by routine hematoxylin and vided to the FDA and the vector was approved for the eosin (H&E) based on morphologic examination by uro- current IND clinical trial. According to a recent HGNC logic pathologists at each institution. Assessment of tumor recommendation, GLIPR1 is the preferred symbol for the for apoptosis [terminal deoxynucleotidyl transferase–medi- RTVP-1 gene. For consistency, we will use Ad5GLIPR1 ated dUTP nick end labeling (TUNEL)] and nuclear p27Kip1 symbol to indicate clinical grade vector for the remaining was done by immunohistochemistry (IHC) on the baseline part of the article. biopsy and RP specimen in a subset of patients. Results from the IHC were compared with results observed in controls Trial design and therapy administration from a tissue microarray (TMA) containing cancer tissues The protocol used in our study was approved by the from RP specimens. Plasma, serum, and urine basic fibro- Biosafety Committees and the Institutional Review Boards blast growth factor and plasma and serum VEGF levels at of the participating institutions of BCM (Veterans Affairs baseline, days 15, 28, and the fourth postoperative day (2 Medical Center, Ben Taub General Hospital, St. Luke’s days at each visit) were done. Blood was drawn at screening Episcopal Hospital), the Recombinant DNA Advisory Com- and days 8, 15, 21, and 28, and the fourth postoperative day mittee of the NIH, and the United States Food and Drug (2 days at each visit) for the following immunologic Administration. The Data and Safety Monitoring Plan was studies by ELISA: ELISPOT assay, NK activity, IL-2, IL-6, applied under an IND by the prostate cancer SPORE pro- IL-12, IFN- g, TGFb, and CD4/CD8 levels. The titer of gram at BCM. serum antibodies to adenovirus was measured at baseline The trial was designed as a conventional phase I trial and day 28 (2 days at each visit). enrolling 3 patients per cohort and evaluating 6 doses of viral particles (vp): 1010,5 1010,1011,5 1011,1012, and Statistical considerations 5 1012 vp. On day 1, a transrectal ultrasound (TRUS) A conventional phase I trial was designed with a sample guided single intraprostate injection of adenoviral GLIPR1 size of 3 to 6 patients for each of 6 doses of virus. The was delivered, followed by RP on day 28. Factors such as maximum tolerated dose (MTD) was the dose for which the tumor size and location determined the injectable volume, incidence of dose limiting toxicities was lesser than 33%. which was to be no more than 2 mL split into 2 separate Although the trial was not expected to have sufficient power injections of 1 mL each into the right and left lobes. An oral, to detect small differences in biomarkers, the preliminary broad-spectrum antibiotic was administered the evening analyses were done using all patients and stratified by dose. before and the morning of the intraprostatic injection and Numerical and graphical descriptive statistics were calcu- continued for 4 days. Following the procedure, the patients lated. Paired t tests or Wilcoxon signed-rank tests were were admitted for a 23-hour observation period. They were employed to evaluate the change in biomarkers from biopsy then followed in the outpatient clinic at days 8, 15, 21, and to RP. The 1-ANOVA or Kruskal–Wallis tests, followed by 28 (2 days at each visit) for a history and physical exam- appropriate multiple comparisons procedures if the overall ination and urinalysis, as well as urine culture and sensi- test was statistically significant at the 5% level of signifi- tivity if necessary. Laboratory work consisting of hematol- cance, were used to compare changes in biological markers ogy and comprehensive metabolic panel survey was mea- between the dose groups to assess differences. sured at baseline and upon completion of therapy before RP. Results

Lymphocyte phenotype by ﬂow cytometry Patient characteristics Automated complete blood counts were done by BCM Nineteen patients were enrolled with a median age of and MD Anderson Cancer Center. Flow cytometric pheno- 64 (range 50–75; Table 1). One additional patient was typing was done after incubating 100 mL of heparinized accrued in the second cohort administered 5 1010 vp blood with the following dual color-labeled antibody pairs: because a patient in this cohort withdrew consent from CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/ participation (although he did undergo therapy followed by HLA-DR, CD4/HLA-DR, CD3/HLA-DR, and CD3/ RP). The routine pathology and toxicity data are available CD56þCD16 (Simultest; Becton Dickinson and company). for the patient who withdrew consent, but correlative After incubation at room temperature for 30 minutes, red studies could not be done after withdrawal of consent.

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Table 1. Clinical and pathologic characteristics A B Patient characteristic Number of patients (%)

Total number of patients 19 Median age (years) 64 (Range 50–75) Clinical stage T1c 9 (47%) T2a 4 (21%) C D T2b 3 (16%) T2c 3 (16%) Gleason score 7 11 (58%) 8 5 (26%) 9 3 (16%) PSA (ng/mL) 10 18 (95%) Figure 2. Cytopathologic examination of prostatectomy specimens 10–20 1 (5%) following AdGLIPR1 therapy. Cytopathic apoptotic effects of AdGLIPR1 10 >20 0 (0%) in prostate cancer RP specimen (1 10 vp) showed in low power ( 100) by H&E staining obtained 4 weeks after a single injection. A, extensive cell Radical prostatectomy pathologic stage death was documented in a large (60%–70%) portion of the RP tissues; pT0 0 (0%) B, high power (400) micrograph of A; C, nonviable tumor tissue pT1-2 12 (63%) juxtaposed to compromised but viable normal prostate epithelium, ﬁ pT3 7 (37%) suggesting tumor cell speci city of cytotoxic effects; D, viable prostate cancer cells in the perineurium surrounding a nerve (in contrast to Nodeþ 2 (11%) nonviable adjacent prostate cancer) suggesting a protective perineural Seminal vesicle invasion 5 (26%) effect. Margin þ 3 (16%) (Fig. 2A–D). The benign prostatic epithelium within the tumor appeared stressed but did not show the cytotoxic effect (Fig. 2C). These morphologic alterations suggest a tumor-speciﬁc cytotoxic effect of AdGLIPR1. Interestingly, a Forty-seven percent of men had a clinical stage of T1c. Fifty- protective perineural effect was observed; the cancer cells eight percent had a Gleason score of 7, and the remainder surrounding a nerve tended to survive the AdGLIPR1- had a Gleason score of 8 or 9. The PSA was 10 ng/mL or less induced cytotoxic effect (Fig. 2D). in 95% of patients.

Safety and feasibility Modulation of tumor tissue biomarkers All 6 doses were feasible with no grade 4 or higher The apoptotic activity was measured by IHC for TUNEL toxicities. Symptomatic toxicities included urinary tract done in 5 patients in the first 2 cohorts (Fig. 3). The results infection (n ¼ 3; grade 3 in 2 men), flu-like syndrome show induction of apoptosis in these patients when pre- (n ¼ 3), grade 1 fever (n ¼ 1), dysuria (n ¼ 1), and treatment biopsy and RP tumor specimens are compared photophobia (n ¼ 1). Asymptomatic laboratory toxicities (Fig. 3A) or when RP specimens were compared with a large n ¼ were grade 1 elevated AST/ALT (n ¼ 1), and elevations of number of individual RP tumor specimens ( 464) PTT (n ¼ 3; 2 were transient, 1 prolonged elevation proven assembled on a TMA (Fig 3B). Further evidence of to be lupus anticoagulant in the 1 1012 cohort). No excess AdGLIPR1 activity was documented in similar studies that Kip1 in postoperative complications was observed. showed increased levels by IHC of nuclear p27 in the same 5 patients from the first 2 cohorts (Fig. 3C). AdGLIPR1 Histopathologic evaluation of radical prostatectomy mediated translocation of p27 to the nucleus in treated specimens patients was shown in comparative analysis of pretreatment Pathologic stage at RP included pT2N0 (n ¼ 12), pT3N0 biopsies with RP specimens (Fig. 3C), or when RP speci- n ¼ (n ¼ 6), and pT3N1 (n ¼ 1), with focal positive margins in 3 mens were compared with RP specimens ( 573) assem- patients (Table 1). No pathologic complete remission bled on a TMA (Fig. 3D). (pCR) was seen, but morphologic evidence of biologic activity was observed, including cytopathic effects and Immunophenotyping analysis of peripheral blood inflammatory infiltrates across all doses. For example, com- lymphocytes þ pared with the pretreatment biopsy, H&E staining from the Cohorts 1 to 3 did not show a clear increase in CD8 RP tissue from the first patient treated with the lowest lymphocytes (Fig. 4A). There was moderate increase of þ dose (1 1010 vp) revealed extensive cytotoxic activity CD8 lymphocytes from 17.9%, 18.1%, and 29.2% in

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Figure 3. Immunohistochemical A B evaluation of prostatectomy specimens following AdGLIPR1. 4.0 2.5 P = 0.0295 10 to Results for 5 patients 1 10 3.5 5 1010 vp for cohort 1, 2 are 2.0 shown. A, apoptotic indices 3.0 (TUNEL) in tumors of patients treated with AdGLIPR1 are 2.5 1.5 significantly increased compared with preoperative biopsies 2.0 Tunel P ¼ Tunel (Wilcoxon signed rank test, 1.0 0.0431, N ¼ 5); B, apoptotic indices 1.5 in patients treated with AdGLIPR1 1.0 (RP) were significantly higher than in 0.5 retrospective (TMA) controls 0.5 (Mann–Whitney Test, P ¼ 0.0295). C, intranuclear p27Kip1 expression 0.0 0.0 in prostate cancer tissues was Bx RP TMA AdGLIPR1 patiens increased after treatment with N = 464 N = 5 AdGLIPR1 (RP) in 4 of 5 patients C D compared with preoperative 9 6 biopsies (Wilcoxon signed rank P = 0.0022 test, P ¼ 0.0656); D, intranuclear 8 p27Kip1 expression in patients 5 7 treated with AdGLIPR1 was significantly higher than in 6 4 retrospective (TMA) controls (Mann–Whitney test, P ¼ 0.0022). 5 3 Box plots were used for 4 2 patients nonparametric data and show the median (line), interquartile range 3 2 P27 Nucl tumor P27 Nucl tumor (the box includes the middle 50% of 2 observations), and whiskers extend 1 out to the farthest points that are not 1 outliers. These plots allow the reader to visually compare 0 0 Bx RP distributions of the variables. TMA AdGLIPR1 patiens N = 573 N = 5

þ cohorts 4 to 6 at pretreatment to 32.4%, 29.9%, and 39.9% DR T cells was already significantly increased to 48.3% þ þ at 2 or 3 weeks after treatment (Fig. 4A). This increase (P ¼ 0.0107), and on day 15, the mean percent of CD8 DR reached statistical significance on days 21 and 28 of cohort T cells was gradually increased to 58.6% (P ¼ 0.0035). It 4(P ¼ 0.0012, P ¼ 0.0440, respectively; unpaired t test; reached a peak at day 21 (60.7% P ¼ 0.0133). After that, the þ þ Fig. 4A, right). percent of DR CD8 T cells remained high until postoper- The HLA-DR marker of activation was used to double label ative day 4 (day 28; 58.2% P ¼ 0.0370, fourth postoperative þ CD4 or CD8 lymphocytes as a relative measure of activated day; 57.8% P ¼ 0.0280; Fig. 4B, bottom right). T cells. There was a moderate increase in the percentage of In cohorts 4, 5, and 6, the posttreatment mean percentage þ þ þ double positive CD8 DR T cells for patients in cohorts 4 to of CD4 T cells positive for the HLA-DR marker of activa- þ 6 (Fig. 4B). The pretreatment mean percentage of CD8 T tion was increased compared with pretreatment levels (Fig. þ þ cells positive for the HLA-DR marker of activation was 4C). The pretreatment mean percentage of CD4 DR T 12.9%, 23.8%, and 20.8% (cohorts 4, 5, and 6). In cohort cells was 8.6%, 10.6%, and 11.6% for cohorts 4, 5, and 6, 4, for days 21 and 28 posttreatment and the fourth postop- respectively, which increased to 16.3%, 22.1%, and 20.3%, þ þ erative day, the mean percent of CD8 DR T cells increased respectively. In cohorts 5 and 6, these increases were sta- by 58.9%, 47.6%, and 34.6%, which were statistically sig- tistically significant (cohort 5; P ¼ 0.0495, cohort 6; nificant (P ¼ 0.001, P ¼ 0.0018, and P ¼ 0.0025, P ¼ 0.0495, Mann–Whitney U test). þ respectively; Fig. 4B, top right). In cohort 5, for days 15, The posttreatment mean percentage of CD3 T cells 21, 28, and the fourth postoperative day, the mean percent- positive for the HLA-DR marker of activation increased þ þ age of CD8 DR T cells significantly increased by 50.3%, compared with pretreatment for cohort 4 (Fig. 4D). The þ þ 62.4%, 63.6%, and 37.9% (P ¼ 0.0036, P ¼ 0.0039, pretreatment mean percentage of CD3 DR T cells was P ¼ 0.0034, and P ¼ 0.0421, respectively; Fig. 4B, bottom 13.3%, 11.8%, and 11.3% (cohort 4, 5, and 6, respectively), þ left). In cohort 6, on day 8, the mean percentage of CD8 which increased to 28.0%, 21.9%, and 31.2%, respectively.

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A Cohorts 1–6, CD8+ cells Cohort 4, CD8+ cells 60 70 60 50 C1 50 40 C2 ** 40 14

* C3 %

% 30 30 15 C4 20 16 C5 20 10 C6 10 Figure 4. Flow cytometry analysis of 0 0 patient samples of circulating Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 peripheral blood lymphocytes at – + + + + indicated time points. Data for each B Cohorts 1 6, CD8 DR cells Cohort 4, CD8 DR cells 70 90 cohort are presented as mean SE. ** ** ** 80 Values that reach statistical 60 * * ** C1 70 signiﬁcance compared with the 50 * ** C2 60 pretreatment levels are indicated by 40 50 14 C3 % % , P 0.05; , P 0.01. A, percent 30 40 15 þ C4 30 16 of CD8 cells at indicated time 20 C5 20 points in cohort 1 to 6 (left). Increase 10 þ C6 10 in percentage of CD8 cells in the 3 0 0 patients in cohort 4 attained Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 statistical signiﬁcance compared Cohort 5, CD8+DR+ cells Cohort 6, CD8+DR+ cells with pretreatment value indicated 90 90 by , P 0.05; , P 0.01 at days 80 80 28 and 21, respectively. Percent of 70 70 þ 60 60 CD8 cells for individual patients in 50 17 50 20 cohort 4 (right). B, percent of %

% þ þ 40 18 40 21 CD8 DR cells in cohorts 1 to 6 at 30 19 30 22 indicated time points during the trial 20 20 (top left). Increase in percentage of 10 10 CD8þDRþ cells that reach 0 0 Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 statistical significance compared with pretreatment value in cohort 4 C Cohorts 1-6, CD4+DR+ Cohort 4, CD4+DR+ (top right), 5 (bottom left), and 6 35 35 (bottom right) indicated. C, percent 30 30 of CD4þDRþ cells at indicated time C1 25 25 points in cohort 1 to 6 (top left). * * C2 20 20 14 Increase in percentage of % C3 % þ þ 15 15 15 C4 CD4 DR cells that reach 16 fi 10 C5 10 statistical signi cance compared 5 C6 5 with pretreatment value in cohort 5 and 6 indicated by , P 0.05. In 0 0 þ þ Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 addition, CD4 DR values are presented for individual patients in + + + + Cohort 5, CD4 DR Cohort 6, CD4 DR cohort 4 (top right) and 5, 6 (bottom 35 35 left and right). D, percent of 30 30 CD3þDRþ cells at indicated time 25 25 points during the trial in cohorts 1-6 20 17 20 20 (left). Increase in percentage of % % 15 18 15 21 CD3þDRþ cells that reach 19 22 10 10 statistical significance compared 5 5 with pretreatment value in cohort 4 0 0 indicated by *, P 0.05. Percent of Pre Day 8 Day15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 CD3þDRþ cells for individual patients in cohort 4 is shown on D Cohort 1-6, CD3+DR+ Cohort 4, CD3+DR+ 50 60 right panel.

50 40 C1 40 C2 30 * 14

C3 % 30 % 15 20 C4 20 16 C5 10 C6 10

0 0 Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4

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These increases attained statistical significance on day 21 of the further development of intraprostatic injection of ade- cohort 4 (P ¼ 0.0495, Mann–Whitney U test) and days 15 noviral vector-delivered GLIPR1 in the perioperative setting and 21 of cohort 6 (P ¼ 0.0495, Mann–Whitney U test). for prostate cancer. Other previously reported trials have established the Analysis of serum cytokines feasibility and biological activity (both local and systemic) The levels of serum TGFb and IFN-g remained of the intraprostatic delivery of favorable disease-modifying unchanged throughout the trial in all cohorts. The level of genes. INGN 201 (Ad-p53), a replication-defective adeno- IL-6 remained at pretreatment levels and was only elevated viral vector that encodes a wild-type p53 gene driven by the at the fourth postoperative day, probably as a result of the cytomegalovirus promoter, was administered in a neoad- surgery, in all patients regardless of treatment groups (data juvant trial of patients with high-risk localized prostate not shown). Serum IL-12 levels also did not show any cancer (14). Of 11 patients with negative baseline p53 significant changes in the majority of the patients and was expression, 10 had expressed p53 and 8 had an increase in less than 200 pg/mL with two exceptions. For patient # 18 apoptosis. To explore the activity of IL-2 expressing adeno- (cohort 5) serum IL-12 levels increased to 426 pg/mL at day virus in prostate cancer, another phase I clinical trial was 8 after treatment that gradually declined to 250 pg/mL on conducted in patients with localized high-risk disease (15). the fourth postoperative day (Fig. 5A). Patient # 20 (cohort An inflammatory response consisting predominantly of þ þ 6) had a moderate increase in serum IL-12 to 239 pg/mL at CD3 CD8 T lymphocytes with areas of tumor necrosis day 15 after treatment that declined gradually to 79 pg/mL was observed. Increases in both g-IFN and IL-4 secreting T on the fourth postoperative day (Fig. 5B). cells were observed. In another small neoadjuvant phase I trial, a DNA–lipid complex encoding the IL-2 gene was Discussion administered intraprostatically (16). Evidence of immune activation was observed, reflected by an increase in T-cell The intraprostatic administration of GLIPR1 tumor sup- infiltration seen in tissue and increased proliferation of pressor gene, expressed by an adenoviral vector, was safe in peripheral blood lymphocytes cocultured with patient men with localized intermediate and high-risk prostate serum. cancer preceding RP. Preliminary evidence of local biolog- A limitation of the neoadjuvant paradigm evaluating ical antitumor activity accompanied by systemic immune biological activity with a novel agent is that the level of responses were observed. Increased levels of tumor cell biological activity that may translate to enhanced objective TUNEL (measuring apoptosis) and nuclear p27Kip1 (a clinical outcomes (progression-free or overall survival) is cyclin-dependent kinase inhibitor) provided corroborative unknown. Therefore, although our trial provides evidence evidence for biological antitumor activity. Systemic induc- of biological activity and systemic immune responses, com- tion of an immune response was observed with an increase plementary information in terms of improved clinical out- þ þ þ in serum IL-12 and circulating CD8 , CD4 , and CD3 T comes in the setting of a randomized trial is necessary. The cells coupled with increased HLA-DR upregulation on these pathologic stages at RP of patients enrolled on our trial are cells, a marker of activation. Indications of a dose response difficult to interpret in the setting of a small phase I trial. A were observed, with serum IL-12 increases in a patient in randomized trial design employing a control arm receiving each of the 2 highest dose cohorts and more robust increases intraprostatic adenovirus vector not carrying GLIPR1 may in peripheral blood circulating activated T lymphocytes in have been more optimal but was considered impractical the higher dose cohorts. In addition, the prolongation of and beyond the scope of available resources. The evaluation PTT in 3 men (1 proven to be lupus anticoagulant) also of tumor tissue correlative studies was not possible in all suggests the generation of a systemic immune response. patients due to resource constraints, although the demon- These data provide proof of concept and suggest a role for stration of biological activity in the 2 lowest dose cohorts

Figure 5. Serum levels of IL-12 for patients in cohort 5 and 6. A, for A Serum IL-12 cohort 5 B Serum IL-12, cohort 6 patient #18 (cohort 5) serum IL-12 450 450 levels increased to 426 pg/mL at 400 400 ﬁrst week after treatment that 350 350 gradually drop to 300 pg/mL at 300 300 week 4 and to 250 pg/mL the fourth 250 #17 250 #20 pg/mL postoperative day. B, patient # 20 pg/mL 200 #18 200 #21 (cohort 6) had moderate increase in 150 #19 #22 serum IL-12 to 239 pg/mL at 150 100 second week after treatment that 100 dropped to 196 pg/mL at week 4 50 50 and to 79 pg/mL the fourth 0 0 Pre Day 8 Day 15 Day 21 Day 28 POD4 Pre Day 8 Day 15 Day 21 Day 28 POD4 postoperative day.

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suggests that activity would probably have been observed in or GLIPR1 hypermethylation may preferentially respond the higher dose cohorts too. Demonstration of an upregu- because GLIPR1 may be expected to be downregulated. lation of GLIPR1 in tumor tissue (following therapy) cor- To conclude, this phase I trial of neoadjuvant intrapro- relating with biological activity may have been desirable but static GLIPR1 tumor suppressor gene delivered by an was considered beyond the scope of this trial. Moreover, this adenoviral vector employed a resource-friendly and small was a phase I trial with the primary goal of showing number of patients and showed biological antitumor feasibility and biological activity, and upregulation of activity and favorable modulation of blood-based bio- GLIPR1 has been shown in previous preclinical studies markers of immune stimulation. Potentially, the biolog- (20, 21). In addition, long-term follow-up with biochemical activity of neoadjuvant adenovirus delivered GLIPR1 ical and clinical recurrence data are not available but will into early tumor tissue may translate into improved also probably be uninterpretable in the absence of a ran- perioperative outcomes as well as activity in more domized design. Although the sparing of perineural tumor advanced settings. In addition, the therapeutic index cells was based on visual interpretation and was not quan- appears excellent, and combinations with other classes tified objectively, a tumor growth promoting microenvi- of tolerable and active biological agents may warrant ronment provided by neurons has been previously exploration, for example, sipuleucel-T or abiraterone ace- described (24). tate (5, 6). The combination of adenovirus delivered The appropriate dose to further develop AdGLIPR1 GLIPR1 with conventional ADT, chemotherapy, and therapy is unclear because no MTD was established. radiotherapy may also warrant exploration. Therefore, the optimal biological dose should be based on further analysis of correlative studies, and the study of Disclosure of Potential Conflicts of Interest larger doses of viral particles may be warranted. Although our study did not evaluate the potentially deleterious T.C. Thompson and C. Ren are coinventors on patents involving therapeutic applications of GLIPR1. These patents are held by Baylor College of effects of transduction of nonmalignant prostate cells, Medicine and licensed to Progression Therapeutics Inc., a private biotech- we have previously shown substantially less preclinical nology start-up. The other authors disclosed no potential conflicts of interest. proapoptotic activity of GLIPR1 delivered to nontrans- formed fibroblasts (20). The simultaneous induction of a Acknowledgments

suppressive immune response was not evaluated, but the The authors thank Dr. Malcolm K. Brenner for critical review of the overall proﬁle strongly supports an immune stimulatory manuscript. response. Although PSA changes were not monitored following the intraprostatic injection, such changes in Grant Support the 4 weeks between the injection and RP are unlikely to be informative because PSA alterations are known to This work was supported by grants from the NIH (SPORE P50-CA58204, SPORE P50-CA140388, and R0150588). occur from prostatic procedures. Circulating tumor cells The costs of publication of this article were defrayed in part by the werenotavailableatthetimeofconductofthistrialbut payment of page charges. This article must therefore be hereby marked are also unlikely to have added information because they advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. are seldom detected in early disease. In addition, biomarkers predictive of response need to be studied; for exam- Received July 22, 2011; revised September 1, 2011; accepted September 8, ple, tumors with p53 mutations, attenuated p53 activities, 2011; published OnlineFirst September 20, 2011.

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www.aacrjournals.org Clin Cancer Res; 17(22) November 15, 2011 OF9

GLIPR1 Tumor Suppressor Gene Expressed by Adenoviral Vector as Neoadjuvant Intraprostatic Injection for Localized Intermediate or High-Risk Prostate Cancer Preceding Radical Prostatectomy

Guru Sonpavde, Timothy C. Thompson, Rajul K. Jain, et al.

Clin Cancer Res Published OnlineFirst September 20, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-11-1899

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